Rat liver mitochondria contain an alpha-ketoglutarate-Fe2 ion, ascorbate-dependent dioxygenase which catalyzes the beta-hydroxylation of 6-N-trimethyllysine. The resulting beta-amino-beta-hydroxy acid undergoes a reverse aldol reaction to give glycine and gamma-butyrobetaine aldehyde. The latter is dehydrogenated to gamma-butyrobetaine, the recognized immediate precursor of carnitine. Crystalline, rabbit liver serine transhydroxymethylase catalyzes the aldol cleavage reaction. The mitochondrial cleavage enzyme will be compared to the supernatant enzyme from the same species for substrate specificy. Interorgan relationships in the formation of carnitine will be investigated.